The present application relates generally to energy reversible acyl-enzymes. In particular, the present application relates to energy reversible acyl-enzymes and the like having a cinnamate or related structure core and an additional reactive group which can be modified to impart new properties to the whole composition.
Various methods to make use of enzyme inactivation have been disclosed. Enzyme inhibition can be used to enhance long term storage of enzymes or to inactivate the enzymes in a pharmaceutical drug. For example, U.S. Pat. No. 5,770,699 describes a process of enzyme inhibition to produce inactivated blood factors. U.S. Pat. No. 5,837,679 discloses a method to extend the half lives of blood factors via a transient modification of blood factors by acylation. U.S. Pat. No. 4,337,244 reports a method of treating venous thrombosis using an inactivated fibrinolytic enzyme.
Enzyme activity can be controlled with inhibitors. Reversible control of enzyme activity with light has been the focus of a number of reports (see U.S. Pat. Nos. 5,114,851 and 5,218,137 to Porter et al.). There are a number of advantages of this concept. Most stricing is the ability to control enzyme activity specifically and rapidly, by exposure to light in vivo or ex vivo.
Porter et al. disclose in U.S. Pat. Nos. 5,114,851 and 5,218,137, the light controllable enzymes are obtained by coupling an enzyme active site amino acid residue to cinnamate (CINN) derivatives to form o-hydroxy cinnamate substituted esters or acyl enzymes, which are inactive. On photolysis, the bond with the active site amino acid residue is cleaved and the active site is exposed. Pizzo et al. [(1986) Ann. N.Y. Acad. Sci. 485: 199-203] reported on the use of an o-hydroxy cinnamate substituted ester, formed by coupling the active site of an enzyme to α-methyl-2-hydroxy-4-diethylaminocinnamic acid.
A subsequent report by Porter, et al. demonstrated the therapeutic potential of the inhibited enzymes. Control of clotting reaction times was concentration dependent and photolysis time dependent. In vivo clotting of abnormal blood vessels in a rabbit model of corneal neovascularization was achieved by injection of the inhibited, “caged” enzymes and application of 366 nm light to the eyes for 25 minutes. See Arroyo et al., Thromb. Haemost. 78, 791-793 (1997). U.S. Pat. Nos. 5,114,851 and 5,218,137, further describe these cinnamate derivatized enzymes and uses thereof.
The usefulness of the inhibited enzymes is a function of the rate and extent of photolysis. Utility is limited if rate of bond cleavage between the cirtamate moiety and the active-site amino acid is slow. Many applications require rapid exposure times, on the order of seconds in most in vitro or typical applications, or at most minutes, in most in vivo applications. Rapid, controlled response times are essential for most clinical applications and are of particular importance with labile enzymes or uses where rapid reaction times are essential, as in clotting.
For example, the formation of an acyl-enzyme between α-chymotrypsin and the p-nitrophenyl ester of p-nitro-trans-cinnamic acid is described by Varfolomeyev, S., et al., [FEBS Lett. 15:118 (1971)]. The bond between the enzyme and the carboxylate group is formed with the hydroxyl group of the serine at the catalytic center of the enzyme [Berezin, I. et al., FEBS Lett. 8:173 (1970)]. The formation of an acyl-enzyme between a-thrombin and the trans-isomer of an ester of o-hydroxy-a-methylcinnamic acid is described by Turner, A., et al., J. Amer. Chem. Soc.109:1274 (1987). The bond between the enzyme and the carboxylate group is formed with the hydroxyl group of the serine-195 at the catalytic center of the enzyme (Turner, A., et al., J. Amer. Chem. Soc. 110:244 (1988)). Exposure of the compound to light led to deacylation. The photoactivation of these enzymes was slow and required light intensities and wavelengths such that appreciable enzyme degradation occurred during photoactivation.
Research in this area has continued. For example, Porter, et al. (Photochem. Photobiol. B 38(1), 61-69 (1997) inserted a biotin derivative on the 2-position of the cinnamate side chain (adjacent to the carboxylate group), which could be bound to avidin, for purposes of purification and immobilization. The modified compound also maintained the ability to be photoactivatable.
Further modifications of the CINN core molecule would be desirable in order to improve inhibited enzyme compositions that can be rapidly and controllably reactivated. For example, it would be desirable to introduce additional sites to react with other molecules for purposes of changing the properties of the acyl-enzyme (such as immobilization or pharmacokinetics), while maintaining the desired properties of photo-activation. The present invention addresses this need.